To produce completely ES cell-derived embryos a clump of ES cells is sandwiched between two tetraploid embryos. For ES cell aggregation chimeras with diploid embryos, one embryo is aggregated with a clump of ES cells. Even in this case, the ES cell internalization is efficient enough to achieve a high level of ES cell contribution.

The benefit of single embryo aggregation is two fold: First, double the number of aggregates can be produced in a single experiment. Second, we have found that the efficiency of germ line transmission of the targeted allele is better than with the sandwich type.

This observation may be explained as follows. When male (XY genotype) ES cells are aggregated with a female (XX genotype) morula, resulting in a phenotypically male, only primordial germ cells derived from the XY ES cells can contribute to the functional gametes. The XX primordial germ cells from the host embryo will not form gametes, therefore favoring the germ line contribution of ES cells containing the desired mutant allele.

This arrangement (XX host morula, XY ES cells) can occur in 50% of the single embryo aggregations, but only in 25% of the sandwich type aggregations.

Flushing of eight-cell stage embryos

Materials:

Dissecting microscope;

Flushing needle (The sharp tip of No. 30 G 1/2 needle is cut off and then rounded using sharpening stone);

Method:

1. Place few rows of KSOMmicrodrops (roughly 3 mm in diameter) into tissue culture dish using syringe (e.g. 3 drops in the first and fourth and 4-5 in the second and third rows), cover with oil.

2. Sterilize aggregation needle by washing in ethanol.

3. Make six or more depressions in each microdrop (leaving a few drops intact for ES cell selection) by pressing the darning needle into the plastic and making slight circular movement. Do not twist the needle. This movement creates a tiny depression with clear smooth wall. It should be deep enough to hold the embryo.

ES cells/embryo aggregation

Materials:

Method:

1. Choose clumps of loosely connected ES cells and transfer them into microdrops (not containing embryos) of aggregation plate for final selection.

2. Select few clumps of ES cells (8 -15 cells in each); place each clump in a depression in the microdrop containing embryos.

3. Pick up the corresponding embryo and place it on the clump, alternatively, if the embryo is already inside the depression, place the clump of cells next to it.

4. Assemble all aggregates in this manner, check the plate, and culture overnight at 37o C, 5% CO2 .

The following morning, the majority of aggregates should have formed blastocysts. We transfer a maximum of 8-10 embryos into each uterine horn of a 2.5 dpc pseudopregnant recipient. Mature CD-1 females are used as pseudopregnant foster mothers and ordered at a weight of 30+ g. In the event of a recipient shortage, it is possible:

to transfer up to 24-26 embryos per recipient;

to culture the aggregates (preferably morulae) for one more day and transfer them into 2.5 day pseudopregnant females;

Dissolve tribromethanol in Tert-amyl alcohol, then add to 200 ml distilled water. Place on a magnetic stirrer until solution is in one phase. Store in brown bottle and keep refrigerated until use. Should be warmed and shaken before use. Dosage is 0.2 ml/10 g body weight .

Method:

The embryo transfer procedure is described in details in many publications, such as the following: